Use of histone acetyltransferase inhibitor amidoximes as anti-proliferative agents

ABSTRACT

Described herein is the use of bisamidoximes compounds for the treatment of HAT malfunction related pathologies.

PRIORITY CLAIM

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/149,892 entitled “USE OF HISTONE ACETYLTRANSFERASE INHIBITORAMIDOXIMES AS ANTIPROLIFERATIVE AGENTS” filed Oct. 2, 2018, which claimspriority to U.S. Provisional Application Ser. No. 62/567,089 entitled“USE OF HISTONE ACETYLTRANSFERASE INHIBITOR AMIDOXIMES ASANTI-PROLIFERATIVE AGENTS” filed Oct. 2, 2017, which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The invention generally relates to use of bisamidoximes as histoneacetyltransferase inhibitors.

2. Description of the Relevant Art

Lung cancer, breast cancer, and colon cancer are three types of commoncancer, which are estimated to be among the top four causes ofcancer-related deaths during 2017. For instance, in 2017, it isestimated that 255,180 new cases of breast cancer will be diagnosed inthe USA (in women and men), and 41,070 breast cancer patients will die,making it the most common cancer diagnosed, and the fourth deadliestcancer.

In the cell, the long double-helical DNA strands are packed into acomplex structure associated with various proteins and RNA. Thisstructure, named chromatin, is folded into several levels. Each level offolding increases the compactness and tightness of the packed DNA.Unfolding of this tightly packed chromatin is important for allDNA-mediated functions, such as producing RNA transcripts(transcription), replicating new DNA strands (replication), andrepairing damages within the DNA. The unfolding of DNA enables theaccessibility of large protein complexes that carry out these tasks. Thebasic repetitive packaging unit of chromatin is a beadlike core particlecomposed of 8 positively charged proteins, called core histones, uponwhich the DNA is wound approximately twice. These beads repeatthemselves millions of times in the chromatin, linked by starches of DNAbetween each consecutive bead, in a basic form described as “beads on astring.” The core particle (the bead), with the two linker DNA segmentsextending from it, is called a nucleosome. The width of this first basiclevel of chromatin fiber is 10 nm (nanometer). The negatively chargedDNA is attracted to the core histones by virtue of their positivecharges and by formation of hydrogen bonds.

Folding the chromatin from the 10 nm fiber to the 30 nm fiber is adynamic and reversible process that is very accurately regulatedtemporally and spatially during embryonic development, during cellulargrowth and differentiation, and in response to environmental cues. The30 nm chromatin fiber is a more compact form that represses theactivation of genes (transcription). This transcription repression bythe 30 nm fiber is crucial for regulating the normal development andactivity of the cell. Some subsets of genes are activated and others aresilenced in a very tightly orchestrated manner. The silencing of proteinproduction of some genes is not less critical for the cell than theactivation of others. For instance, cancer can develop when some genes,known as oncogenes, stop being repressed.

One of the major mechanisms for unfolding and folding chromatin is byacetylation and deacetylation of the tails of the core histones,respectively. Deacetylation and consequently folding of chromatin iscarried out by a group of enzymes named histone deacetylases (HDACs).These HDACs are involved in removing the acetyl groups from the corehistones and enabling a higher attraction between the more positivelycharged core histones and the DNA, which result in an increase of thecompaction. There are four classes of HDACs and 11 known HDACs in thezinc-dependent HDAC classes I, II and IV. The activity of HDACs isreversed by another group of enzymes named Histone acetyltransferases(HATs), which are involved in unfolding chromatin. HATs act by adding anacetyl group to the core histones and consequently neutralizing thepositive charge of the histones and alleviating the strong interactionbetween the negatively charged DNA and the less positively chargedhistones.

FIG. 1 shows how the steady-state of histone acetylation is maintainedby opposing activities of histone acetyltransferases and deacetylases.Acetylation is the transfer of the acetyl moiety from an acetyl coenzymeA donor to the side chain of a lysine residue in an acceptor protein.This is an example of a post-translational modification of a protein,since this chemical change happens in the mature protein and not as partof its synthesis. The steady state of histone acetylation is maintainedby the opposing activities of two groups of enzymes, HATs, that add theacetyl group and HDACs that remove it.

In recent years, many research groups have focused their attention onthe discovery of drugs that inhibit HDACs and can inhibit the growth ofmalignant cells. Some of these drugs are already in clinical use.Compounds that inhibit HATs were also discovered, and they, as well,caused a selective growth inhibition and death of malignant cells.Furthermore, recently there have been several reports that the dietarycompound curcumin inhibits the activity of the histone acetyltransferasep300 and prevents heart failure in animals. Potential HAT malfunctionrelated pathology has been demonstrated in a variety of diseases such asAlzheimer, diabetes, hyperlipidaemia, as well as in asthma, and COPD,making HAT inhibitors sought-after compounds. Despite the progress madein understanding the structure of HATs, such as p300, their enzymaticmechanism and the way the HATs are inhibited is essentially unclear.Also, several HAT inhibitors demonstrated reactivity, instability, lowpotency, and lack of selectivity between HAT subtypes and other enzymes.

There is a continuing need for new anti-cancer compounds, since theexisting compounds are not curing all cases of cancer and have numerousnegative side effects. Furthermore, many of the currently usedchemotherapeutic agents result in a drug resistance response inpatients, in which case the patient's tumors stop responding to thetreatment. Histone modifiers, such as HDAC inhibitors and HATinhibitors, are sought-after compounds, since many of them specificallytarget malignant cells by either suppressing oncogenes or inducing theexpression of tumor-suppressor genes. Some of these compounds arealready in clinical use, especially HDAC inhibitors. However, there isstill a need for anti-cancer compounds exhibiting superior effectivenesswhile minimizing side effects associated with many anti-cancercompounds.

SUMMARY OF THE INVENTION

In an embodiment, a method of treating cancer in a subject comprisesadministering to a subject who would benefit from such treatment atherapeutically effective amount of a pharmaceutical compositioncomprising one or more bisamidoximes.

In one embodiment, one or more of the bisamidoximes have the structure:

where W: is —(CH₂)₂—; —(CH₂)₃—; —(CH₂)₄—; or —(CH₂)₃—N(CH₃)—(CH₂)₃—; andwhere Y is H, C₁-C₆ alkyl, or halogen. In one embodiment, Y is methyl.

In one embodiment, one or more of the bisamidoximes are selected fromthe group of compounds consisting of JJMB 5, JJMB 7, and JJMB 9:

In one embodiment, the pharmaceutical composition comprises two or morebisamidoximes. The pharmaceutical composition may comprise JJMB5 andJJMB 9, JJMB5 and JJMB7, or JJMB7 and JJMB9.

In one embodiment, the cancer is breast cancer, lung cancer, or coloncancer.

In one embodiment, a method of treating cancer in a subject comprisingadministering to a subject who would benefit from such treatment atherapeutically effective amount of a pharmaceutical compositioncomprising an anticancer agent and one or more bisamidoximes. In oneembodiment, the anticancer agent is cisplatin.

In one embodiment, a method of inhibiting histone acetyltransferases(HATs) in a biomedical application, comprising applying an effectiveamount of a composition comprising one or more bisamidoximes to abiomedical application that includes HATs.

BRIEF DESCRIPTION OF THE DRAWINGS

Advantages of the present invention will become apparent to thoseskilled in the art with the benefit of the following detaileddescription of embodiments and upon reference to the accompanyingdrawings in which:

FIG. 1 depicts a schematic diagram of how the steady-state of histoneacetylation is maintained by opposing activities of histoneacetyltransferases and deacetylases;

FIG. 2 depicts a graph of mammary tumor volumes in JJMB7 treated anduntreated control BALB/c mice;

FIG. 3 is a graph depicting the effect of compound JJMB9 on breastcancer tumor volume;

FIG. 4 depicts the daily weight of mice during treatment with compoundJJMB9;

FIG. 5 depicts the effect of compound JJMB9 on Mouse Liver Microsomes;

FIG. 6 depicts the effect of JJMB 5, 6, 7, 9 and garcinol on cellularcore histone acetylation;

FIG. 7A depicts the percentage of standardized acetylation on H3K9;

FIG. 7B depicts the percentage of standardized acetylation on H4K5;

FIG. 8 depicts a schematic diagram of the inhibition of HDACs by TSA andhow this induces hyperacetylation of core histones;

FIG. 9A shows how the amidoxime JJMB 5 reverses TSA inducedhyperacetylation;

FIG. 9B shows how the amidoxime JJMB 6 reverses TSA inducedhyperacetylation;

FIG. 9C shows how the amidoxime JJMB 7 reverses TSA inducedhyperacetylation;

FIG. 9D shows how the amidoxime JJMB 9 reverses TSA inducedhyperacetylation;

FIG. 9E shows how garcinol reverses TSA induced hyperacetylation;

FIG. 10A depicts the results from a test studying the effects ofreversing the TSA effect by the amidoxime JJMB5;

FIG. 10B depicts the results from a test studying the effects ofreversing the TSA effect by the amidoxime JJMB6;

FIG. 10C depicts the results from a test studying the effects ofreversing the TSA effect by the amidoxime JJMB9;

FIG. 10D depicts the results from a test studying the effects ofreversing the TSA effect by the amidoxime JJMB7;

FIG. 10E depicts the results from a test studying the effects ofreversing the TSA effect by garcinol;

FIG. 11 depicts a diagram showing the experimental design of the invitro HAT assay in the presence and absence of inhibitor;

FIG. 12 depicts results of the in vitro P300 inhibition assay;

FIG. 13 depicts the percentage of standardized acetylation of H4K5; and

FIG. 14 depicts the results of the in vitro GCN5 inhibition assay.

While the invention may be susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and will herein be described in detail. Thedrawings may not be to scale. It should be understood, however, that thedrawings and detailed description thereto are not intended to limit theinvention to the particular form disclosed, but to the contrary, theintention is to cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the present invention as definedby the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood the present invention is not limited toparticular devices or methods, which may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include singular and pluralreferents unless the content clearly dictates otherwise. Furthermore,the word “may” is used throughout this application in a permissive sense(i.e., having the potential to, being able to), not in a mandatory sense(i.e., must). The term “include,” and derivations thereof, mean“including, but not limited to.” The term “coupled” means directly orindirectly connected.

The terms used throughout this specification generally have theirordinary meanings in the art, within the context of the invention, andin the specific context where each term is used. Certain terms arediscussed below, or elsewhere in the specification, to provideadditional guidance to the practitioner in describing the devices andmethods of the invention and how to make and use them. It will beappreciated that the same thing can be said in more than one way.Consequently, alternative language and synonyms may be used for any oneor more of the terms discussed herein, nor is any special significanceto be placed upon whether or not a term is elaborated or discussed ingreater detail herein. Synonyms for certain terms are provided. Arecital of one or more synonyms does not exclude the use of othersynonyms. The use of examples anywhere in this specification, includingexamples of any terms discussed herein, is illustrative only, and in noway limits the scope and meaning of the invention or of any exemplifiedterm.

As used herein, the term “cancer” refers to a cellular disordercharacterized by uncontrolled or dysregulated cell proliferation,decreased cellular differentiation, inappropriate ability to invadesurrounding tissue, and/or ability to establish new growth at ectopicsites. The term “cancer” includes, but is not limited to, solid tumorsand bloodborne tumors. The term “cancer” encompasses diseases of skin,tissues, organs, bone, cartilage, blood, and vessels. The term “cancer”further encompasses primary and metastatic cancers.

The terms “cells” or “groups of cells” as used herein furtherencompasses cultured cells that have been explanted from a body ortissue and that have been maintained in vitro in a cell culture system.Examples of such cells include “primary cell” cultures. Primary cellsare those cells that are explanted directly from a donor organism ortissue. Primary cells may typically be capable of undergoing a limitednumber of divisions in culture, but they generally do not continue togrow and eventually senesce and die.

Further examples of isolated cells include “secondary cell” cultures.Secondary cells are those cells that are explanted directly from a donororganism or tissue and that are maintained and propagated in culture fora protracted period of time, typically exceeding that of primary cells.Often times, secondary cells may be propagated in vitro for up to asmany as 100 generations or more. Secondary cells are typically notimmortalized however, and eventually undergo senescence. The number ofcell divisions that secondary cells may undergo is related to theirdegree of differentiation. More terminally differentiated cells undergofewer cell divisions and senesce early. Less well-differentiated cells,such as embryonic fibroblasts and cells that have begun to undergoneoplastic transformation, typically have a higher generation potentialand can undergo a greater number of divisions.

Yet further examples of isolated cells include “immortalized cells.”Immortalized cells may typically be maintained and propagated in vitroindefinitely as long as the correct culture conditions are maintained.Immortalized cell lines are commonly referred to in the art as“transformed cells.” The growth properties of such cells are altered.Typically, such cells have undergone one or more genotypic changes, suchas, for example point mutations, aneuploidy or other chromosomalalterations. Immortalized cells may or may not be cancerous ormalignant. Non-malignant transformed cells typically exhibit one or moreof several properties when grown in vitro. Non-limiting examples of thephenotypic properties exhibited by non-malignant transformed cellsinclude anchorage-dependent growth, growth factor dependence, andgrowth-arrest under conditions of nutritional deficiency. Furthermore,while transformed cells are generally not as highly differentiated astheir primary or secondary counterparts, they nonetheless typicallyretain at least a subset of the morphological and biochemical propertiesof the cell type from which they are derived. Finally, non-malignantcells exhibit a growth property known in the art as “contactinhibition.” Typically, such cells will continue to grow and divide invitro when plated at low density. When the density of cells issufficient so that a “monolayer” of cells has formed (i.e., the bordersof adjacent cells are substantially touching), growth inhibitory signalspass between the cells, the cells exit the cell cycle and ceasedividing. Such “contact inhibited” cells are frequently coupled by gapjunctions. Loss of contact inhibition is a widely regarded sign thatcells have become cancerous or oncogenic. Such cells do not stopdividing when they form a monolayer in culture. Rather, they continue todivide and pile up on top of one another in “foci”. It is generally wellaccepted by ordinary practitioners of the art that cells that form fociin culture are tumor cells.

As used herein, the term “neoplastic transformation” or “oncogenictransformation,” generally refers to a proliferative disorder of cellscharacterized by one or more of several cellular changes. Such cellularchanges are manifested by cells that have become, or are on the way tobecoming, cancerous or malignant. Characteristics of cells that haveundergone neoplastic transformation are well known to ordinarypractitioners of the art and may include, but are not limited to, lossof contact inhibition, escape from control mechanisms, loss of GJIC,increased growth potential, increased growth rate, the ability to formcolonies in soft agar, alterations in the cell surface, alterations inthe expression of certain protein or gene markers, karyotypicabnormalities, aneuploidy, morphological and biochemical deviations fromthe norm, and other attributes that confer the ability of the cell orgroup of cells to invade, metastasize, and kill. Neoplastictransformation may be induced, at least in part, by exposure of a cellor group of cells to radiation, or to one or more oncogenic agents suchas certain viruses or carcinogens. A “carcinogen” as used herein,generally refers to a substance that increases the likelihood that acell or group of cells begins the process of neoplastic transformation.Carcinogens may include genotoxic agents, also known in the art as“mutagens”, and non-genotoxic agents, which induce neoplasms bynon-genomic mechanisms.

The term “apoptosis,” as used herein, generally refers to amorphologically distinct form of programmed cell death that is importantin the normal development and maintenance of multicellular organisms.Dysregulation of apoptosis can take the form of inappropriatesuppression of cell death, as occurs in the development of cancers, orin a failure to control the extent of cell death, as is believed tooccur in acquired immunodeficiency and certain neurodegenerativedisorders. Apoptosis is an active process in which cells induce theirself-destruction in response to specific cell death signals or in theabsence of cell survival signals. It is distinct from necrosis, which iscell death occurring as a result of severe injurious changes in theenvironment. Apoptosis of a cell can be characterized at least by therapid condensation of the cell with collapse of the nucleus butpreservation of membranes; or, cleavage of nuclear DNA at the linkerregions between nucleosomes to produce fragments which can be easilyvisualized by agarose gel electrophoresis as a characteristic ladderpattern. Cells undergoing apoptosis exhibit a characteristic series ofmorphological changes including mitochondrial membrane swelling andrupture, leakage of cytosolic contents into the surrounding area, andinflammation in tissues. The pattern of events occurring duringapoptosis is orderly and includes; cell shrinkage; appearance ofbubble-like blebs on their surface; degradation of chromatin (DNA in acomplex with protein and RNA) in their nucleus; mitochondrial ruptureand release of cytochrome c into the cytosol; breakage of the cell intosmall, membrane-wrapped, fragments (commonly referred to as “apoptoticbodies” or “corpses”); exposure of phosphatidylserine on the outerleaflet of the cell membrane; and recruitment of phagocytic cells likemacrophages and dendritic cells which then engulf the cell fragments.

Various pathologies occur due to a defective or aberrant regulation ofapoptosis in the affected cells of an organism. For example, defectsthat result in a decreased level of apoptosis in a tissue as compared tothe normal level required to maintain the steady-state of the tissue canpromote an abnormal increase of the amount of cells in a tissue. Thishas been observed in various cancers, where the formation of tumorsoccurs because the cells are not dying at their normal rate.

The terms “reducing,” “inhibiting” and “ameliorating,” as used herein,when used in the context of modulating a pathological or disease state,generally refers to the prevention and/or reduction of at least aportion of the negative consequences of the disease state. When used inthe context of an adverse side effect associated with the administrationof a drug to a subject, the term(s) generally refer to a net reductionin the severity or seriousness of said adverse side effects.

As used herein, the term “systemically,” when used in the context of aphysiological parameter, generally refers to a parameter that affectsthe entire body of a subject, or to a particular body system.

As used herein the terms “administration,” “administering,” or the like,when used in the context of providing a pharmaceutical or nutraceuticalcomposition to a subject generally refers to providing to the subjectone or more pharmaceutical, “over-the-counter” (OTC) or nutraceuticalcompositions in combination with an appropriate delivery vehicle by anymeans such that the administered compound achieves one or more of theintended biological effects for which the compound was administered. Byway of non-limiting example, a composition may be administeredparenteral, subcutaneous, intravenous, intracoronary, rectal,intramuscular, intra-peritoneal, transdermal, or buccal routes ofdelivery. Alternatively, or concurrently, administration may be by theoral route. The dosage administered will be dependent upon the age,health, weight, and/or disease state of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and/or the natureof the effect desired. The dosage of pharmacologically active compoundthat is administered will be dependent upon multiple factors, such asthe age, health, weight, and/or disease state of the recipient,concurrent treatments, if any, the frequency of treatment, and/or thenature and magnitude of the biological effect that is desired.

As used herein, the term “treat” in the context of animals generallyrefers to an action taken by a caregiver that involves substantiallyinhibiting, slowing or reversing the progression of a disease, disorderor condition, substantially ameliorating clinical symptoms of a diseasedisorder or condition, or substantially preventing the appearance ofclinical symptoms of a disease, disorder or condition.

As used herein, terms such as “pharmaceutical composition,”“pharmaceutical formulation,” “pharmaceutical preparation,” or the like,generally refer to formulations that are adapted to deliver a prescribeddosage of one or more pharmacologically active compounds to a cell, agroup of cells, an organ or tissue, an animal or a human. Methods ofincorporating pharmacologically active compounds into pharmaceuticalpreparations are widely known in the art. The determination of anappropriate prescribed dosage of a pharmacologically active compound toinclude in a pharmaceutical composition in order to achieve a desiredbiological outcome is within the skill level of an ordinary practitionerof the art. A pharmaceutical composition may be provided assustained-release or timed-release formulations. Such formulations mayrelease a bolus of a compound from the formulation at a desired time, ormay ensure a relatively constant amount of the compound present in thedosage is released over a given period of time. Terms such as “sustainedrelease” or “timed release” and the like are widely used in thepharmaceutical arts and are readily understood by a practitioner ofordinary skill in the art. Pharmaceutical preparations may be preparedas solids, semi-solids, gels, hydrogels, liquids, solutions,suspensions, emulsions, aerosols, powders, or combinations thereof.Included in a pharmaceutical preparation may be one or more carriers,preservatives, flavorings, excipients, coatings, stabilizers, binders,nanoparticles, solvents and/or auxiliaries that are, typically,pharmacologically inert. It will be readily appreciated by an ordinarypractitioner of the art that, pharmaceutical compositions, formulationsand preparations may include pharmaceutically acceptable salts ofcompounds. It will further be appreciated by an ordinary practitioner ofthe art that the term also encompasses those pharmaceutical compositionsthat contain an admixture of two or more pharmacologically activecompounds, such compounds being administered, for example, as acombination therapy.

The term “pharmaceutically acceptable salts” includes salts preparedfrom by reacting pharmaceutically acceptable non-toxic bases or acids,including inorganic or organic bases, with inorganic or organic acids.Pharmaceutically acceptable salts may include salts derived frominorganic bases include aluminum, ammonium, calcium, copper, ferric,ferrous, lithium, magnesium, manganic salts, manganous, potassium,sodium, zinc, etc. Examples include the ammonium, calcium, magnesium,potassium, and sodium salts. Salts derived from pharmaceuticallyacceptable organic non-toxic bases include salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines, and basic ion exchange resins, suchas arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine,diethylamine, 2-dibenzylethylenediamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, etc.

As used herein the terms “subject” generally refers to a mammal, and inparticular to a human.

Terms such as “in need of treatment,” “in need thereof,” “benefit fromsuch treatment,” and the like, when used in the context of a subjectbeing administered a pharmacologically active composition, generallyrefers to a judgment made by an appropriate healthcare provider that anindividual or animal requires or will benefit from a specified treatmentor medical intervention. Such judgments may be made based on a varietyof factors that are in the realm of expertise of healthcare providers,but include knowledge that the individual or animal is ill, will be ill,or is at risk of becoming ill, as the result of a condition that may beameliorated or treated with the specified medical intervention.

By “therapeutically effective amount” is meant an amount of a drug orpharmaceutical composition that will elicit at least one desiredbiological or physiological response of a cell, a tissue, a system,animal or human that is being sought by a researcher, veterinarian,physician or other caregiver.

The term “pharmacologically inert,” as used herein, generally refers toa compound, additive, binder, vehicle, and the like, that issubstantially free of any pharmacologic or “drug-like” activity.

In an embodiment, a method of treating cancer in a subject comprisingadministering to a subject who would benefit from such treatment atherapeutically effective amount of a pharmaceutical compositioncomprising a bisamidoxime. As described herein, bisamidoximes have beenshown to be effective against a plurality of types of cancer, includingbut not limited to breast, lung, and colon cancer.

In an embodiment, a method of treating cancer in a subject comprisingadministering to a subject who would benefit from such treatment atherapeutically effective amount of a pharmaceutical compositioncomprising one, two or more bisamidoximes.

In some embodiment, bisamidoximes have the structure:

where W: is —(CH₂)₂—; —(CH₂)₃—; —(CH₂)₄—; or —(CH₂)₃—N(CH₃)—(CH₂)₃—; andwhere Y is H, C₁-C₆ alkyl, or halogen. In a preferred embodiment,bisamidoximes have the structure above where Y is methyl. Specificexamples of bisamidoximes include, but are not limited to JJMB5, JJMB 6,JJMB 7, and JJMB 9:

In some embodiments, using a combination of two or more bisamidoximesproduces a synergistic effect that enhances the effectiveness of thepharmaceutical composition. Synergistic effects have been observed incombinations of: JJMB5 and JJMB 9; JJMB5 and JJMB7; and JJMB7 and JJMB9.

In another embodiment, a method of treating cancer in a subjectcomprising administering to a subject who would benefit from suchtreatment a therapeutically effective amount of a pharmaceuticalcomposition comprising an anticancer agent and one or morebisamidoximes. As described herein, a combination of a known anticanceragent and one or more bisamidoximes have been shown to be effectiveagainst a plurality of types of cancer, including but not limited tobreast and colon cancer.

As used herein, the term “anticancer agent” refers to any agent that isadministered to a subject with cancer for purposes of treating thecancer. Use of the subject bisamidoximes may be particularlyadvantageous and may enhance the effectiveness of the anticancer agent.Non-limiting examples of anticancer agents include topoisomerase Iinhibitors (e.g., irinotecan, topotecan, camptothecin and analogs ormetabolites thereof, and doxorubicin); topoisomerase II inhibitors(e.g., etoposide, teniposide, and daunorubicin); alkylating agents(e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide,carmustine, lomustine, semustine, streptozocin, decarbazine,methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators(e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators andfree radical generators such as bleomycin; nucleoside mimetics (e.g.,5-fluorouracil, capecitibine, gemcitabine, fludarabine, cytarabine,mercaptopurine, thioguanine, pentostatin, and hydroxyurea); cellreplication disrupters (e.g., paclitaxel, docetaxel, and relatedanalogs; vincristine, vinblastin, and related analogs; thalidomide andrelated analogs (e.g., CC-5013 and CC-4047)); protein tyrosine kinaseinhibitors (e.g., imatinib mesylate and gefitinib); antibodies whichbind to proteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Nine novel bisamidoximes were tested for their potential to inhibitgrowth of malignant cell-lines in culture conditions. The bisamidoximeswere synthesized according to the procedure set forth in the paper toJohnson et al. “Bisamidoximes: Synthesis and Complexation withIron(III)”, Aust. J. Chem. 2007, 60, 685-690, which is incorporatedherein by reference.

Table 1 summarizes the results of the testing of the nine novelbisamidoximes. In Table 1, the various compounds listed were tested forinduction of death in various malignant human cell lines and werecompared to the commercial HAT inhibitor garcinol (0.2-500 μM).Specifically, Table 1 describes the summary of the growth inhibitoryeffect (in μM) of nine novel bisamidoximes on six malignant cell lines,as measured by MTS assay. MTS assay is a colorimetric based cytotoxicityassay that determines the number of viable cells. The numbers in thetable represent Growth Inhibition GI₅₀ (±standard deviation), which isthe concentration of the drug where 50% of the cells are killed.Garcinol, a known HAT inhibitor, was used as a positive control. Thecell lines used were: HCT-116 (colorectal carcinoma), DU-145 (prostatecarcinoma), SK-OV-3 (ovarian adenocarcinoma), HLF-a (lung epidermoidcarcinoma), MCF-7 & MDA-MB-231 (breast adenocarcinoma) and NHDF (normalhuman dermal fibroblasts). The cells were treated with compounds JJMB1-9 for 48 hrs (MCF-7 for 72 hrs) at various concentrations ranging from0.2 μM to 500 μM. One-way ANOVA was performed to assess the differencesbetween the groups. Differences in means between control and treatmentgroups were analyzed by post-hoc Dunnett's test, p<0.05. Data isreported as the mean±SD, n=3. N-No inhibition; *—represents results fromhighly purified and more potent batches of the compounds. The compoundstested (JJMB5, JJMB6, JJMB7, and JJMB9) could efficiently kill severalmalignant cell-lines in concentration range of micromolars. (See Table1). Furthermore the compounds tested did not kill normal human dermalfibroblast cells (NHDF) at these concentrations, making them goodcandidates for continuing biochemical, pharmacological, and eventuallymedical studies. From these test it is clear that the compounds affectedseveral cells lines of the three major types of cancers: breast, lung,and colon.

Since the various potent amidoximes demonstrated a distinct pattern ofcytotoxicity by killing different cell lines, it suggested that each ofthese compounds has a different mechanism of action. To furtherunderstand the mechanism of action of the four active compounds (JJMB5,JJMB6, JJMB7, and JJMB9), we performed several experiments that aresummarized in Table 2. All the assays were performed on HCT-116 (acolorectal carcinoma), since all of the four specific potent drugs couldinduce death of these cells. The assays demonstrated that all thecompounds induced apoptosis of cells (programed cell death), and all ofthem reduced the acetylation levels of core histones H3K9 and H4K5 inHCT-116 cells, which suggested that these compounds are HAT inhibitors.JJMB9 indeed inhibited the activity of a purified recombinant HAT—p300in vitro (in a tube). Another HAT, GCN5, was not inhibited by any of thecompounds. JJMB5, JJM6 and JJMB9 could reverse the activity of an HDACinhibitor (TSA); thus the hyperacetylation induced by TSA could bereversed by these compounds.

TABLE 1 Induction of death in malignant human cell lines bybisamidoximes (JJMB 5, 6, 7, and 9), and the commercial HAT inhibitorgarcinol (0.2-500 μM) Cell Line JJMB5 JJMB6 JJMB7 JJMB9 Garcinol Colon(HCT-116)  8.5* ± 2.5 17 ± 4 6.2* ± 2   16* ± 3.4 39 ± 8 Lung (HLF-A)335 ± 4 N 275 ± 3 40 ± 7 27 ± 4 Breast (MCF-7), 72 hours 33.2* ± 6.0 N N8.5* ± 2.5 55 ± 7 Breast (MDA-MB-231) 125 ± 4 N  85 ± 3 N 44 ± 4Prostate (DU-145) N N N N 55 ± 7 Ovarian (SKOV-3) N N N N 43 ± 5 Normal(NHDF) N N N N N

TABLE 2 Comparison of various characteristics of the bisamidoximes thatspecifically killed malignant cells in culture ACTIVITY JJMB5 JJMB6JJMB7 JJMB9 Induction of apoptosis in HCT-116 cells + + + + Inhibitionof histones H3K9 acetylation + + + + in HCT-116 cells Inhibition ofhistones H4K5 acetylation + + + + in HCT-116 cells Inhibition ofhistones H3K27 acetylation + + − + in HCT-116 cells Inhibition of theHAT p300 in vitro − − − + Inhibition of the HAT GCN5 in vitro − − − −Reversion of HDAC-inhibitor-induced + + − + hyperacetylation Inhibitionof core histone acetylation in + + − + vivo prior to onset of apoptosisInduction of G1/S arrest − + + + Induction of G2/M arrest + − − −This is another indication that the three compounds, JJMB5, JJMB6, andJJMB9 are HAT inhibitors. However, bisamidoxime JJMB7 could not reversethe TSA-induced hyperacetylation, indicating that it is not a HATinhibitor. In accord with this notion, JJMB7 did not demonstrateinhibition of core histones acetylation in vivo prior to the onset ofapoptosis, as opposed to JJMB5, JJMB6 and JJMB9. This result indicatedthat JJMB7 induces an indirect decrease of acetylation, not byinhibiting HATs, but by some alternative mechanism. The overall picturethat emerges from the data presented in Tables 1 and 2 suggests thateach of the four bisamidoximes that showed cell-death induction activityhas a different mode of action.

Though the compounds induced apoptosis (programmed cell death) ofseveral malignant lines, due to the fact that two different breastcancer cell lines, one estrogen receptor positive and the other estrogenreceptor negative, were sensitive to the bisamidoximes, and due to thevery high incidence of breast cancer, our approach in the experimentalwork was to focus on breast cancer. However, it should be understoodthat the results set forth herein are applicable to other forms ofcancer, particularly colon cancer and lung cancer.

The efficacy of killing malignant cells in mice and slowing down thetumors or eradicating them can be indicative to these compounds'efficacy in humans. To check if the bisamidoximes are potent in mice,breast cancer cells were identified that could be implanted in femalemice mammary fat pads. The identified cells were tested to see if theyare sensitive to bisamidoximes in vitro (in tube/cell culture).

It was found that the murine mammary cancer cell line 4T1 (and also theEpH4 cells) was highly sensitive to three bisamidoximes that killedhuman breast cell lines (JJMB5, and JJMB9). The growth inhibition 50%(GI₅₀) of the various drugs on 4T1 cells were as follows: JJMB5—6.3±2.3μM; JJMB7—14.7±6.3 μM; and JJMB9—33.3±2.4 μM. At the next stage, weneeded to determine whether the mice would tolerate the drugs, or inother words, what would be the maximum tolerated dose (MTD) of each ofthe bisamidoximes. The three bisamidoximes JJMB5, JJMB7, and JJMB9, aswell as a control group of only the vehicle (the solvent used todissolve the drugs) were injected intraperitoneally (IP) to mice. Theprotocol included injection of the desired concentration of bisamidoximeover 5 consecutive days, two days of break, and another cycle of 5successive days of injections. It was determined that the maximumtolerated doses in mice (BALB/c strain) were as follows: JJMB5—0.26mg/kg; JJMB7—1.56 mg/kg; and JJMB9—0.78 mg/kg.

The bisamidoxime JJMB7 was studied to see if the compound can lower therate of tumor growth and the number and size of metastases in mice.JJMB7, was found to significantly reduce the mammary carcinoma tumorvolume when injected IP. FIG. 2 depicts a graph of mammary tumor volumesin JJMB7 treated and untreated control BALB/c mice. In the experiment,six-week-old BALB/c mice were implanted with 10,000 4T1 breast carcinomacells in the mammary fat pad (MFP). The next day, 10 mice were injectedIP with the maximum tolerated dose (MTD) of 1.56 mg/kg of JJMB7, and 10control mice were injected with the vehicle alone. JJMB7 wasadministered for five consecutive days, followed by a two day break, andanother five consecutive days of injections. Mice were weighed every dayand observed for signs of pain and discomfort. Tumors were visible 7days after implantation, and tumor dimensions were measured, and volumeswere calculated until day-24, when mice were euthanized. A One-WayRepeated Measures ANOVA was performed to determine a statisticalsignificance (p≤0.05).

In parallel to the in vivo testing, tests were performed to study theefficacy of various combinations of the bisamidoximes on killing humanmalignant cancer cells in culture. Since each of our four compoundsbeing tested have a different mode of action, each of the compounds mostlikely have a different molecular target in the cells.

The results of various tests of combinations of bisamidoximes onmalignant cancer cell lines are summarized in Table 3. Combinationstudies performed on human breast cancer cell line MCF-7 and human coloncancer cell line HCT 116 were measured by MTS assay. The cells weretreated with novel bisamidoximes JJMB 5, 7, and 9, for 48 hours (72hours for MCF-7), individually or in combination. Growth Inhibition(GI₅₀) was measured. *- Indicates a significant difference when thedrugs were used in combination compared to when the drugs were usedindividually as determined by one-way ANOVA, p≤0.05.

TABLE 3 Combination studies of novel bisamidoximes on malignant celllines Cell Line Amidoxime Growth Inhibition (GI₅₀) in uM MCF-7 JJMB528.2 ± 7.1 JJMB9  5.3 ± 2.7 JJMB5 & JJMB9  5.1 ± 3.3* HCT-116 JJMB5 20.4± 3.6 JJMB9 20.9 ± 3.5 JJMB5 & JJMB9  9.4 ± 2.2* JJMB5 22.4 ± 1.1 JJMB715.5 ± 5.6 JJMB5 & JJMB7  5.7 ± 1.4* JJMB7 25.2 ± 1.7 JJMB9 22.4 ± 4.4JJMB7 & JJMB9  7.2 ± 1.5* 4T1 JJMB5  7.8 ± 3.2 JJMB9 26.1 ± 5.9 JJMB5 &JJMB9  3.4 ± 0.8 JJMB5  8.2 ± 0.1 JJMB7  7.1 ± 0.8 JJMB5 & JJMB7  3.2 ±0.7* JJMB7  8.0 ± 0.6 JJMB9 30.2 ± 2.0 JJMB7 & JJMB9  4.2 ± 0.6*

The results described in Table 3 demonstrate that combinations of JJMB5and JJMB9, JJMB5 and JJMB7, or JJMB7 and JJMB9 were more efficient inkilling the colon cancer cells HCT-116 (lower GI_(50S)) than using eachof the bisamidoximes alone. The same combinations were also moreefficient in inducing the death of the murine mammary carcinoma cells4T1.

The results of various tests of combinations of bisamidoximes withcisplatin on malignant cancer cell lines are summarized in Table 4.

TABLE 4 Combination studies of cisplatin and novel bisamidoximes onhuman malignant cell lines Cell Line Amidoxime Growth Inhibition (GI50)in μM MCF-7 JJMB5 33.2 ± 6.0 Cisplatin 48.5 ± 6.5 JJMB5 & Cisplatin 17.2 ± 3.8* JJMB9  8.5 ± 2.5 Cisplatin 40.8 ± 2.6 JJMB9 & Cisplatin   5 ± 1.2* HCT-116 JJMB5 19.3 ± 2.4 Cisplatin 68.6 ± 8.5 JJMB5 &Cisplatin  7.9 ± 0.5* JJMB7 16.2 ± 2.0 Cisplatin  73 ± 9.0 JJMB7 &Cisplatin  6.2 ± 1.0* JJMB9  16 ± 3.4 Cisplatin 66.3 ± 8.4 JJMB9 &Cisplatin  8.1 ± 1.0*Combination studies of bisamidoximes and cisplatin were performed onhuman breast cancer cell line MCF-7, human colon cancer cell lineHCT-116, and murine mammary malignant cell lines measured by MTS assay.The cells were treated with novel bisamidoximes JJMB 5, 7, and 9, for 48hours (72 hours for MCF-7), individually or in combination withcisplatin. Growth Inhibition (GI₅₀) was measured. *—Indicates asignificant difference when the drugs were used in combination withcisplatin compared to when the drugs were used individually asdetermined by one-way ANOVA, p≤0.05.

The results summarized in Table 4 show that the combination of thecommonly used chemotherapeutic agent cisplatin and JJMB5, and cisplatinand JJMB9, were more efficient in killing the human breast cancer cellsMCF-7. The combination of JJMB5, JJMB7, or JJMB9 with cisplatin was alsomore efficient in killing the colon carcinoma cells HCT-116. Inaddition, it appears that the bisamidoximes JJMB5, JJM7, and JJMB9 weremore efficient individually than the commercially available and widelyused cisplatin.

FIG. 3 shows that compound JJMB9 significantly reduced the breast cancertumor volume up to day 21 since the implantation of the malignant cells(mouse mammary carcinoma). The murine mammary carcinoma 4T1 cells wereimplanted in the mammary fat pad of BALB/c female mice (10,000 cells permice) and a day later we started a protocol of two 5-day cycles (with 2days break) of chemotherapy delivered IP. The amidoximes were deliveredat their MTD level—JJMB5 with 0.26 mg/kg, and JJMB9 with 0.78 mg/kg.One-Way Repeated-Measure ANNOVA was performed to show the significance(p≤0.05, n=10).

FIG. 4 shows the daily weight of mice during this experiment, indicatingthat though the drug-injected mice lost some weight during the firstdays of injection, they later bounced back to the normal weight rangeshown by the untreated control mice group. The untreated mice were alsotransplanted with 4T1 cells, but they were not injected the vehicle orone of the drugs.

FIG. 5 demonstrates our study with Mouse Liver Microsomes which revealedthe t½ of JJMB9 to be 13 minutes.

The amidoximes described herein contain an amidoxime group,characteristic of some HDAC inhibitors. However, these compounds alsohave a six-carbon ring connected by a linker, characteristic of some HATinhibitors. To test if the amidoximes induced reduction of acetylationor increased acetylation levels of core histones, we performed Westernblot analysis against acetylated H3K9 and acetylated H4K5 on the wholecell lysates after treating HCT-116 cells with amidoximes for 24 hoursat various concentrations. The experiment was repeated three times.Histone H3 lysine 9 (H3K9) and histone H4 lysine 5 (H4K5) are wellstudied and important sites involved mainly in transcriptionalregulation.

FIG. 6 depicts the effect of JJMB 5, 6, 7, 9 and garcinol on cellularcore histone acetylation. The acetylation levels of core histones inHCT-116 cells were measured following treatment for 24 hours atconcentrations ranging from 0.04 μM to 80 μM. HCT-116 cells (Panels a-e)were incubated with the amidoximes for 24 hours and then lysed. Westernblot analysis was conducted by using antibodies against Ac-H3K9 andAc-H4K5. Coomassie staining of the SDS-PAGE was done to demonstrateequal loading of the protein. a) Significant reduction in acetylationlevels is seen at a concentration of 20 μM and higher after JJMB 5treatment. b) JJMB 6 treatment resulted in reduction of acetylationlevels at 30 μM on H3K9 and H4K5. c) JJMB 7 treatment resulted inreduced acetylation levels at 40 μM and higher concentrations. d) JJMB 9treatment resulted in complete inhibition of acetylation levels seen at40 μM. e) Garcinol, used as a positive control, also showed reduced corehistone acetylation seen at 25 and 50 μM. f) No change in acetylationlevels was observed in DU-145 cells treated with JJMB 9. DMSO was usedas a vehicle control and applied at concentrations used at the highestamidoxime treatment, in percentage ranging from 0.04%-0.08%. Theseresults suggest that, amidoximes that induced death of HCT-116 cellsreduced the core histone acetylation in vivo.

To determine if induction of death in cancer cells is correlated withreduced core histone acetylation, this experiment was repeated withDU-145 cells prostate carcinoma. By MTS cell viability assay, wedemonstrated that DU-145 cells were not killed by JJMB 9 (see Table 1).Therefore, we treated DU-145 cells for 24 hours with increasing doses ofJJMB 9 and Western blot analysis was performed to test the acetylationlevels of core histones with antibodies against Ac-H3K9 and Ac-H4K5. Theresults showed that JJMB 9 did not inhibit the levels of acetylation onAc-H3K9 and Ac-H4K5 in DU-145 cells (FIG. 6 , f). These results suggestthat the inhibition of core histone acetylation occurred only in thecells that were killed by amidoximes.

FIG. 7 depicts the percentage of standardized acetylation on H3K9 (FIG.7A) and H4K5 (FIG. 7B) which was calculated by normalizing theacetylation levels to the O.D of the core histones obtained bydensitometry of coomassie staining. This graph represents the means of 3individual experiments. One-way ANOVA was performed to determine thedifferences between the groups. Pair-wise comparison was done to findout the significance between control group and treatment groups. Theseresults were confirmed by Kruskal Wallis and Mann-U-Whitneynon-parametric tests. Levels of significance were designated as *p<0.05.Data is reported as the mean±SEM (n=3).

The reduced core histone acetylation in cells treated with amidoximescan be explained mainly in three ways:

-   1) The amidoximes inhibit histone acetyltransferases (HATs) directly    or indirectly.-   2) The amidoximes activate histone deacetylases (HDACs) directly or    indirectly.-   3) The amidoximes increase the expression levels of HDAC genes or    decreases the expression levels of HAT genes in the cell.

TSA (trichostatin A) is an HDAC inhibitor that inhibits class I and IIHDACs but not class III HDACs. The inhibition of HDACs causes thehyperacetylation of lysine residues on the core histone tails. In theprevious experiment, we demonstrated that amidoximes caused reduction incore histone acetylation in HCT-116 cells (see FIGS. 6 and 7 ). Theprimary reason for the reduced acetylation levels caused by amidoximescould be due to the inhibition of HATs. Our hypothesis is that, cellstreated with a combination of HDAC inhibitor, TSA and amidoximes wouldreverse the TSA induced hyperacetylation. FIG. 8 depicts a schematicdiagram of the inhibition of HDACs by TSA and how this induceshyperacetylation of core histones. Inhibition of HATs on the other handcauses hypoacetylation.

To determine if amidoximes can reverse the TSA induced-hyperacetylation,we treated cells with each amidoxime alone, TSA alone and each amidoximetogether with TSA for 24 hours and cell lysates were extracted. Westernblot analysis was performed using antibodies against acetylated H3K9 andacetylated H4K5. DMSO treated cells (Lane 1, FIG. 9 a-e ) showed basalacetylation of core histones, followed by a dramatic increase inacetylation levels upon TSA treatment (Lane 2, FIG. 9 a-e ). Thepositive controls, amidoxime alone (Lane 6, FIG. 9 a-d ) and garcinolalone (Lane 5, FIG. 9 e ) showed inhibition of acetylation levelscompared to DMSO treated cells (Lane 1, FIG. 9 a-e ). The co-treatmentof HCT-116 cells with the positive control, garcinol at 25 μM and TSAresulted in the reduced acetylation levels of core histones whencompared to TSA alone treated cells (Lane 2, FIG. 9 e ). Theco-treatment of HCT-116 cells with amidoximes JJMB 5 (20 μM and 40 μM)or JJMB 6 (30 μM and 45 μM) or JJMB 9 (20 μM and 40 μM) and TSA showedreduced acetylation levels compared to TSA alone treated cells (Lane 2,FIG. 9 a-e ). These results suggest that JJMB 5, 6, 9 and garcinoleffectively reversed the TSA induced hyperacetylation at higherconcentrations, whereas JJMB 7 did not reverse the TSA inducedhyperacetylation (FIG. 9 ). Results also support the possibility thatamidoximes JJMB 5, 6 & 9 are HAT inhibitors but the possibility ofhistone hypoacetylation could be an indirect effect as well.

The effect of concomitant treatment with TSA and amidoximes on cellsurvival was studied. From the previous experiment, we learned thattreatment of HCT-116 cells with the combination of TSA and amidoximesreversed the TSA induced hyperacetylation in most cases. Amidoximesreverse the HDAC inhibitor, TSA induced hyperacetylation. HCT-116 cellswere treated with amidoximes alone (at the indicated concentrations),TSA alone (0.3 μM) and amidoximes concomitantly with TSA for 24 hoursand cell lysates were extracted. Western blot analysis was performedusing the antibodies against Ac-H3K9 and Ac-H4K5. JJMB 5, 6 and 9effectively reversed TSA induced hyperacetylation whereas JJMB 7 did notaffect the TSA induced hyperacetylation. Garcinol also opposed the TSAinduced hyperacetylation at 25 μM.

Acetylation of core histones is generally involved in activation of geneexpression whereas deacetylation of core histones is generally involvedin gene repression. Our hypothesis was that reversing the TSA effect byamidoximes would also block the cell death induced by TSA or by theamidoximes. In other words, reversing the TSA effect by amidoximes wouldbring the global acetylation and gene expression levels to normal steadystate levels and therefore the cells would survive. To determine if theamidoximes can block the TSA induced cell death, HCT-116 cells weretreated with amidoximes alone (at the indicated concentrations in FIGS.10A-E), TSA alone (0.3 μM) or TSA incubated along with JJMB 5, 6, 7 and9 at various concentrations and MTS cell viability assay was performedafter 24 hours. One-way ANOVA was performed to assess the differencesbetween groups. Differences in means between TSA treated and TSA alongwith amidoximes groups were analyzed by post-hoc Dunnett's test,*p<0.05. Values are mean±SD, n=3. MTS assay was performed to determinecell viability. The results revealed that TSA alone and amidoximes aloneeffectively induced cell death in HCT-116 cells when compared to DMSOtreated cells (FIG. 10 ). When we compared the cells treated with TSAalone and TSA incubated along with amidoximes JJMB 5, 6, 7, 9 andgarcinol, no significant blocking of cell death was observed in any ofthe compounds tested except for JJMB 5 at 8 μM with TSA. These resultsindicate that the reason for the cell death is not simply a change inthe global acetylation steady state of core histones but something morecomplex.

JJMB 9 is a p300 Inhibitor

The previous experiments have shown the inhibition of core histoneacetylation in HCT-116 cells when treated with amidoximes (see FIG. 6 ).This was also supported by the fact that JJMB 5, 6, and 9 reversed theTSA induced hyperacetylation suggesting that inhibition of core histoneacetylation levels could be due to the inhibition of histoneacetyltransferases (HATs) (see FIG. 9 ). This prompted us to examine ifthese amidoximes can inhibit the HATs in vitro. To test this hypothesis,we selected p300, a HAT from p300/CBP family which is capable ofacetylating free and nucleosomal histones both in vitro and in vivo. TheHAT inhibitory activity was assayed using full length human recombinantp300, purified core histones from chicken erythrocytes and acetyl coA asthe substrates. FIG. 11 depicts a diagram showing the experimentaldesign of the in vitro HAT assay in the presence and absence ofinhibitor. Western blot analysis was performed to determine if theamidoximes inhibited the p300-dependent acetylation on H3K9 and H4K5.FIG. 12 depicts results of the in vitro P300 inhibition assay. In vitrohistone acetyltransferase inhibition (HAT) assay was performed usingacetyl CoA (72 μM) and purified core histones as substrates for the HATp300 (Calbiochem) in the absence or presence of increased concentrationsof JJMB 5, 6, 7 and 9 (8 μM-1000 DMSO was used as a vehicle control(lane 2). Garcinol, a known HAT p300 inhibitor was used as a positivecontrol. Reaction mixtures were run on 15% SDS-PAGE and Western blot wasperformed using antibodies against Ac-H4K5 and Ac-H3K9. Lane 1represents the basal acetylation levels of H3K9 and H4K5. Increase inthe acetylation levels was observed in lane 2 upon p300 addition (FIG.12 ).

FIG. 13 depicts the percentage of standardized acetylation of H4K5. p300dependent acetylation activity of histone H4K5 was inhibited by JJMB 9in a dose dependent manner as compared to the DMSO control. One-wayANOVA was performed to determine the significance of the differencesbetween the groups. Post-hoc Dunnett's test was done to find out thesignificance between control group and treatment groups. Levels ofsignificance based on post-hoc Dunnett's test were designated as *p<0.05& **p<0.01. Data is reported as the mean±SEM of three experiments.

As expected garcinol inhibited the p300-dependent acetylation on H3K9 at20 μM and at 5 μM on H4K5 compared to DMSO. JJMB 5, 6 and 7 didn'tinhibit p300-dependent acetylation in vitro. In contrast, JJMB 9 wasfound to inhibit p300 activity in a dose dependent manner with an IC50of 40 μM (FIGS. 12 and 13 ). The p300 mediated acetylation was almostcompletely inhibited at 1000 μM by JJMB 9 compared to DMSO (*p<0.05)(FIG. 13 ). Garcinol completely inhibited the p300-dependent acetylationat 100 μM whereas JJMB 9 inhibited at 1000 μM with a ten-folddifference. This suggests that garcinol is potent inhibitor of p300compared to JJMB 9 (FIG. 12 ).

Amidoximes Did Not Inhibit the GCN5 In Vitro

Since amidoximes JJMB 5, 6 and 7 did not inhibit the p300 HAT in vitro,we tested the possibility that these amidoximes inhibit another type ofHAT. To test this hypothesis, we selected GCN5, GNAT family HAT, whichis capable of acetylating histones both in vitro and in vivo. The HATinhibitory activity was assayed using full length mouse recombinant GCN5(purified in our lab) and purified core histones and acetyl coA as thesubstrates. Western blot analysis was performed to determine if theamidoximes inhibit the GCN5-dependent acetylation of Ac-H3K14. For GCN5inhibition assay, we used only anti Ac-H3K14 antibody since rGCN5predominantly acetylates histone H3 at lysine 14.

FIG. 14 depicts the results of the in vitro GCN5 inhibition assay. Thisassay was performed using acetyl CoA (72 μM) and purified core histones(2 μg) as substrates for the HAT GCN5 (purified) in the absence orpresence of increased concentrations of JJMB 5, 6, 7 and 9 (8 μM-500μM). Anacardic acid, a known HAT GCN5 inhibitor was used as a positivecontrol. Reaction mixtures were run on 15% SDS-PAGE and western blot wasperformed using antibodies against Ac-H3K14. JJMB 5, 6, 7 and 9 did notinhibit the acetylation activity of GCN5 whereas anacardic acidinhibited the GCN5 acetylation activity in a dose dependent manner. Lane1 represents the basal acetylation levels of H3K9 and H4K5. Increase inthe acetylation levels was observed in lane 2 upon GCN5 addition. Asexpected anacardic acid inhibited the GCN5-dependent acetylation at 40μM. The results revealed that JJMB 5, 6, 7 and 9 did not inhibitGCN5-dependent acetylation in vitro.

In this patent, certain U.S. patents, U.S. patent applications, andother materials (e.g., articles) have been incorporated by reference.The text of such U.S. patents, U.S. patent applications, and othermaterials is, however, only incorporated by reference to the extent thatno conflict exists between such text and the other statements anddrawings set forth herein. In the event of such conflict, then any suchconflicting text in such incorporated by reference U.S. patents, U.S.patent applications, and other materials is specifically notincorporated by reference in this patent.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as examples of embodiments. Elements and materials maybe substituted for those illustrated and described herein, parts andprocesses may be reversed, and certain features of the invention may beutilized independently, all as would be apparent to one skilled in theart after having the benefit of this description of the invention.Changes may be made in the elements described herein without departingfrom the spirit and scope of the invention as described in the followingclaims.

What is claimed is:
 1. A method of inhibiting histone acetyltransferases(HATs) in a HAT malfunction related pathology, comprising administeringto a subject who has a medical condition associated with the HATmalfunction related pathology an effective amount of a compositioncomprising one or more bisamidoximes, wherein the one or morebisamidoximes are selected from the group of compounds consisting ofJJMB 5, JJMB6, and JJMB 9


2. The method of claim 1, wherein the pharmaceutical compositioncomprises two or more bisamidoximes.
 3. The method of claim 1, whereinthe HAT malfunction related pathology is Alzheimer's disease.
 4. Themethod of claim 1, wherein the HAT malfunction related pathology isdiabetes mellitus.
 5. The method of claim 1, wherein the HAT malfunctionrelated pathology is hyperlipidaemia.
 6. The method of claim 1, whereinthe HAT malfunction related pathology is asthma.
 7. The method of claim1, wherein the HAT malfunction related pathology is COPD.